Method and article for detecting the fertile period

ABSTRACT

A NEW AND RAPID TEST METHOD IS PROVIDED FOR PREDICTING AND DETECTING THE FERTILE PERIOD OF THE FEMALE. THE METHOD INVOLVES A SIMPLE RAPID TEST FOR ALKALINE PHOSPHATASE ACTIVITY IN THE SALIVA OF THE FEMALE. IN THE METHOD THE FEMALE WETS A NOVEL TEST IMPLEMENT WITH HER SALIVA AND THE TEST IMPLEMENT WILL TURN A DETECTABLE BLUE COLOR IN ABOUT ONE MINUTE IF AN AMOUNT OF ALKALINE PHOSPHATASE IS PRESENT IN THE SALIVA SUFFICIENT TO CAUSE THE COLOR CHANGE. THIS AMOUNT OF ALKALINE PHOSPHATASE IS CONINCIDENT WITH THE ONSET OF THE FERTILE PERIOD OF THE FEMALE, AND EXTENDS FOR THE DURATION OF THE FERTILE PERIOD. THE NOVEL DRY TEST IMPLEMENT CONSISTS OF A DRY BIBULOUS MATERIAL WHICH HAS BEEN IMPREGNATED BEFORE DRYING WITH AN INDOXYL PHOSPHATE AND A BUFFER.

United States Patent O 3,699,005 METHOD AND ARTICLE FOR DETECTING THEFERTILE PERIOD Raymond 0. Foster, 1202 Clara St., Joliet, II]. 60435 NoDrawing. Continuation-impart of abandoned application Ser. No. 597,547,Nov. 29, 1966. This application Aug. 26, 1970, Ser. No. 67,258

Int. Cl. G01n 31/14 U.S. Cl. 195-103.5 R 6 Claims ABSTRACT OF THEDISCLOSURE A new and rapid test method is provided for predicting anddetecting the fertile period of the female. The method involves a simplerapid test for alkaline phosphatase activity in the saliva of thefemale. In the method the female wets a novel test implement with hersaliva .and the test implement will turn a detectable blue color inabout one minute if an amount of alkaline phosphatase is present in thesaliva sulficient to cause the color change. This amount of alkalinephosphatase is coincident with the onset of the fertile period of thefemale, and extends for the duration of the fertile period. The noveldry test implement consists of a dry bibulous material which has beenimpregnated before drying with an indoxyl phosphate and a buffer.

CROSS REFERENCE This application is a continuation-in-part of my earlierU.'S. application Ser. No. 597,547 which was filed Nov. 29, 1966 and nowabandoned.

BACKGROUND OF THE INVENTION There has been much interest in the past aswell as presently in being able to predict and detect the period of timeduring which the female is fertile, i.e., is capable of conceiving anoffspring. The fertile period occurs during ovulation, when the ovum hasdeparted the ovary and is still alive and in a position to befertilized.

The importance is readily apparent of being able to predict and detectthe fertile period, both to those who wish to take advantage of thefertile period and those who wish to avoid it. However, prior to thepresent invention, the methods and means available for such predictionand detection have failed to provide the degree of convenience andaccuracy desired. Attempts to calculate said period by reference to theonset of menses are speculative and beset with inaccuracy because thehuman female, for instance, may ovulate at varying and unpredictabletimes. On the other hand, mechanical methods such as by taking andrecording temperatures, while possibly more accurate, are distinctlyinconvenient and may require considerable skill in observing andinterpreting the data.

It has been found that alkaline phosphatase, an enzyme found in thesaliva (or other body secretions, e.g., cervical mucus) varies inconcentration during the female menstrual cycle, and has a relativelyhigher concentration during the first part of the females menstrualcycle and at the time of ovulation.

It is known in the chemical art that indigo, a strong "ice blue dye, isreadily formed by the coupling of two indoxyl radicals. (Fieser andFieser, Organic Chemistry, 1944, p. 872.) It is also known in the artthat the presence of the enzyme, alkaline phosphatase, can be detectedin tissue sections by a staining technique which depends upon depositionof indigo in crystalline form at the spot where the enzyme is localizedin the tissue. (Holt, Proc. Roy. Soc., B 148, 1958, pp. -168.)

The procedure described by Holt is a tissue staining technique requiringnot only specialized equipment to perform the procedure, but also highlytrained personnel to conduct the test and read the results. Holtsprocedure is a non-quantitative laboratory method which requiresprolonged incubation, the addition of an oxidizing agent such as aferricyanide to increase the rate of depositions, and also must begraded microscopically.

In the face of the foregoing discouraging information in the prior art,I have, nevertheless, discovered a new and rapid test method forpredicting and detecting the fertile period of the female quiteaccurately by a simple test which can be conducted by anyone regardlessof technical skill. The test comprises contacting saliva of a femalewith a novel test implement which is a piece of dry, bibulous materialwhich has been impregnated before drying with an indoxyl phosphate and abuffer. If alkaline phosphatase is present in the saliva in significantamount, as I have found it to be just prior to and during the fertileperiod, then the implement will turn a detectable blue color in aboutone minute after wetting with saliva. The implement will remain in itsoriginal colorless state if the female is not approaching nor within thefertile period.

SUMMARY OF THE INVENTION This invention relates to the discovery thatthe fertile period of the female may be predicted accurately bycontacting the females saliva with a novel test implement, and to anovel analytical test implement useful in detecting the fertile periodof the female which is coincident with an increase in certain enzymeactivity.

Further in accordance with the present invention, there is now provideda test implement useful in detecting an increase in alkaline phosphataseactivity which is coincident with the fertile period of the female whichcomprises a bibulous material impregnated with a non-toxic buffercapable of maintaining the pH at an alkaline level in the range of about10.0 to about 10.3 and an indicator characterized by the formula R-O-POwherein R is an indoxyl radical which upon cleavage of the ester bond bysaid alkaline phosphatase will form or develop a compound having areadily discernible color.

While any physiologically acceptable indoxyl compound having theabove-specified formula and properties can be employed as the indicator,it has been found that indoxyl phosphate and five-bromo-indoxylphosphate are particularly advantageous.

The cleavage of the ester bond in the aforesaid indoxyl compoundsreleases the indoxyl radical, or substituted indoxyl radical and, in thepresence of oxygen or air two indoxyl radicals combine to form indigo,or a substituted indigo, a dark blue dye. In the practice of thisinvention this reaction normally takes place in a short period of time,such as about thirty seconds to about one minute. The degree of blueformed is relative to the amount of the alkaline phosphatase present.

The presence of phosphate ions in the saliva and those formed by thecleavage of the indicator member tends to inhibit the action of theenzyme, alkaline phosphatase. But, it has now been found that the actionof the enzyme can be significantly increased by including in the testtape a compound capable of accepting and tying up phosphate ions. Thecompound should be inert with respect to the indicator and the buffer.Magnesium salts, e.g., the sulfate and chloride, have been found to besuitable.

The great advantage and convenience of this oral test is at onceapparent. The female simply touches the test paper to her tongue to wetit, and waits a few minutes to see if a color change develops on thetest paper. The re sult is easily observed, and does not require therecording and interpretation of data over a long period of time. Eachtest is complete per se. The test is reliable and simple as well asbeing convenient and relatively inexpensive.

This method and implement is also highly useful to the clinician indetecting pathological conditions because positive tests at a knownnon-fertile period are a clear indication of unusual and perhapspathological conditions.

DESCRIPTION OF THE PREFERRED EMBODIMENT Describing in more detail thetest implement or test paper, the test paper is preferably bibulouspaper, for example, an absorbent and chemically pure grade of filterpaper or the like. Cloth strips or porous and absorbent wood strips mayalso be employed. The test paper is impregnated with a solution of theactive ingredients and is then subjected to drying to evaporate thesolvents and leave the active ingredients on the paper.

In one representative formula, when indoxyl phosphate is the indicator,1.5 grams of sodium alginate is dissolved in 100 cc. distilled water.Five grams of tris (hydroxy methyl) aminomethane, 2 grams of sodiumcarbonate and 500 milligrams of magnesium sulfate are dissolved in 100cc. of distilled water. Two grams of indoxyl phosphate is dissolved in40 cc. of distilled water.

The three solutions are combined with the resultant solution having a pHof about 10.0 to about 10.3. The large sheets of chemically pure filterpaper are impregnated with the mixed solution and air dried.

Five bromo-indoxyl phosphate can be substituted for the indoxylphosphate in the above representative formula to serve as the indicator.The concentration of the indicator can be varied within a range of 500to 2500' milligrams depending upon the degree of final color desired.

It has been found on detailed study that a representative test paper,which is a piece of standard filter paper about inch wide by 1 inchlong, will absorb on the average about 25 mg. of saliva on astandardized wetting time of about 5 seconds. In a normal female duringher nonfertile period, this amount of saliva will contain an amount ofalkaline phosphatase that is not significant and can be detected onlywith much difiiculty. Special techniques and formulations are needed andthe development of a readable color takes so long as to be totallyimpractical. For example, time in excess of ten minutes is required andby then the test paper will have dried, shutting off the chemicalreaction that develops the color. Thus, any amount of alkalinephosphatase lower than about 2.5 to 3.0 units of activity in the 25 mg.sample will not give an easily discernible color change. (A unit ofalkaline phosphatase activity is defined as a microgram of phenol formedper ml. of sample in 69 minutes at 37 C.)

A typical test paper made as described above will have absorbed about102 gamma of indoxyl phosphate after drying.

In one typical female it was found that a daily com- 4 parison of thebasal body temperature with a chemical analysis of the amount ofalkaline phosphatase ina 25 mg. sample of saliva showed excellentcoordination between the drop in temperature normally associated withthe immediate pre-ovulatory and early ovulatory stage and a rise inalkaline phosphatase activity to amount above the detectable level, forthis patient, about 3.0 units of activity. Thus an activity high enoughto be easily read on the subject test implement coincided with theimmediate pre-ovulatory and ovulatory phase. This patient would knowfrom a color change on my test implement that she was entering or was inher fertile period.

The test papers or test tapes so prepared have been used in clinicalstudies to determine the accuracy of the test tapes in detecting thefertile period as evidenced by color changes caused by the saliva of thefemale. When compared to the previous standard, namely, the (basal bodytemperature) thermal shift response, the foregoing test tapes give asignificant correlation.

There is thus provided a convenient and accurate test method and testpaper for detecting the fertile period by a simple test on saliva. Thetest papers above described, after contact with saliva during thefertile period, will detect an increase in alkaline phosphatase activityby developing a readily discernible color after but a short period oftime. If there has been no significant increase in alkaline phosphataseabove the amount normally present during a non-fertile period, the tapedoes not develop the readily discernible color upon contact with saliva.

While the foregoing test method and test tape are obviously of primaryimportance to the human female, it is apparent that the test isapplicable to other female animals, and can be of considerableimportance.

The response of the test implement may also be demonstrated bycontacting other closely related body fluid, namely, cervical mucouswith the test implement. The foregoing invention has been described indetail, but it will be apparent to those skilled in the art that certainsubstitutions can be made among the ingredients employed herein without,however, departing from the spirit of the invention. All suchsubstitutions are considered to be included herein, provided they fallwithin the scope of the appended claims.

I claim:

1. The method of detecting the fertile period of a female by use of asystem which changes color coincident with the onset of the fertileperiod, which comprises wetting with saliva from a female, a drybibulous test implement impregnated with an effective amount of anindicator selected from the group consisting of indoxyl phosphate andS-bromo-indoxyl phosphate, said amount being sufficient to produce acolor change when the alkaline phosphatase concentration of the femalessaliva is at the elevated level coincident with the onset of the fertileperiod, and a non-toxic buffer which maintains the pH in the range fromabout 10.0 to about 10.3, observing the test implement for a colorchange within about one minute after wetting with the saliva, andpredicting said fertile period based on the presence or absence of acolor change.

2. The method of claim 1 wherein said indicator is indoxyl phosphate.

3. The method of claim 1 wherein said indicator is 5- bromo-indoxylphosphate.

4. A test implement useful in detecting the fertile period of a femaleby use of a system which changes color coincident with the onset of thefertile period and adapted to detect the presence of an elevated levelof alkaline phosphatase activity in saliva upon wetting said implementwith saliva and observing the color change, which consists of a dry,bibulous material impregnated with an effective amount of an indicatorselected from the group consisting of indoxyl phosphate andS-bromoindoxyl phosphate, said amount being sufficient to produce acolor change when the alkaline phosphatase concentra- 6 tion is at theelevated level coincident with the onset of the fertile period, and anon-toxic bufier which maintains OTHER REFERENCES the pH in the rangefrom about 10.0 to about 10.3. Holt, Nature, VOL 169, 271473 (1952) 5.The test implement of claim 4 wherein said indicator Holt Nature, Vol. 1PP- 1012-1014 13 mdoxyl Phosphate- 5 Goldschmidt et al., BiologicalAbstracts, vol. 33,

6 The test implement of claim 4 wherein said indicaparagraph 40384(1959). tor 1s S-bromo-mdoxyl phosphate.

ALVIN E. TANENHOLTZ, Primary Examiner References Cited UNITED STATESPATENTS 10 195 99 l- X-R- 2,905,594 9/1959 Morris 195-l03.5

